This invention relates to the epidermal growth factor related proteins, polynucleotides encoding these proteins and methods for using these proteins. The cellular machinery involved in mitogenesis is complex, and not yet fully understood. In general, receptors present on the cell surface bind growth factors, resulting in an activated receptor. In particular, tyrosine kinase receptors (TKRs) are endowed with intrinsic tyrosine kinase activity. The activated receptors, in turn, phosphorylate intracellular substrates. These phosphorylated substrates are responsible for a series of events that leads to cell division. This process is generally referred to as xe2x80x9cmitogenic signal transduction.xe2x80x9d The molecular machinery involved in this process is considered to be the xe2x80x9cmitogenic signaling pathway.xe2x80x9d
Growth factors and hormones exert pleiotropic effects on cellular functions, including mitogenic stimulation and modulation of differentiation and metabolism (Ullrich, et al. Cell 61:203-212 (1990); Aaronson, S. A. Science 254: 1146-1153 (1991)). In many cases, these actions are mediated by the interaction of growth factors with cell surface tyrosine kinase receptors (TKRs), which results in enhanced receptor catalytic activity and tyrosine phosphorylation of intracellular substrates (Ullrich, et al., supra, Aaronson, supra). Knowledge of the nature of these second messenger systems is still scanty, although some molecules which associate and/or are tyrosine phosphorylated by TKRs have been identified. These include the xcex3 isozyme of phospholipase C (PLC-7) (Margolis, et al. Call 57: 1101-1107 (1989), Meinsenhelder, et al. Cell 57: 1109-1122 (1989) and Wahl, et al. Mol. Cell. Biol. 9: 2934-2943 (1989)); the p21ras GTPase activating protein (GAP) (Molloy, et al. Nature 342: 711-714 (1989), Kaplan, et al. Cell 61:125-133 (1990), and Kazlauskas, et al. Science 247: 1578-1581 (1990)); the raf serine-threonine kinase (Morrison, et al. Proc. Natl. Acad. Sci. USA 85: 8855-8859 (1988), and Morrison, et al. Cell 58: 649-657 (1989)); the p85 subunit of the phosphatidylinositol 3-kinase (Ptdlns-3K); (Coughlin, et al. Science 243: 1191-1194 (1989) Kazlauskas, et al. Cell 58: 1121-1133 (1989), Varticovski, et al. Nature 342: 699-702 (1989), Ruderman, et al. Proc. Natl. Acad. Sci. USA 87: 1411-1415 (1990), Escobedo, et al. Cell 65: 75-82 (1991), Skolnik, et al. Cell 65: 83-90 (1991), Otsu, et al. Cell 65: 91-104 (1991)) and some cytoplasmic tyrosine kinases (Gould, et al. Mol. Cell. Biol. 8: 3345-3356 (1988); Kypta, et al. Cell 62: 481-492 (1990)). These signaling molecules are thought to mediate at least in part the mitogenic effects of TKRs (Ullrich, et al. supra; Aaronson, supra).
However, the Epidermal growth factor (EGF) receptor (EGFR) does not appear to efficiently interact with known second messenger systems (Fazioli, et al. Mol. Cell. Biol. 11: 2040-2048 (1991); Segatto, et al. Mol. Cell. Biol. 11: 3191-3202 (1991)). Thus, there is need to ascertain the mechanism by which the EGFR functions in mitogenesis, and a particular need to identify and characterize the substrate (if any) of the EGFR.
Errors which occur in the mitogenic signaling pathway, such as alterations in one or more elements of that pathway, are implicated in malignant transformation and cancer. It is believed that in at least some malignancies, interference with such abnormal mitogenic signal transduction could cause the cells to revert to normal phenotype.
In addition, reagents useful in identifying molecular components of the mitogenic signaling pathway find utility as tumor markers for therapeutic, diagnostic, and prognostic purposes. Furthermore, identification of how such components differ from normal components in malignant tissue would be of significant value in understanding and treating such malignancies.
EGFR, a 170 kDa transmembrane glycoprotein protein with intrinsic tyrosine kinase activity, which binds EGF family of peptides, plays an important role in controlling cell proliferation and differentiation as was shown by Ullrich et al. supra. The EGFR possesses three functional domains that include extracellular, transmembrane and cytoplasmic. Ligand binding to the extacellular domain of EGFR leads to dimerization and activation of the receptor""s intrinsic tyrosine kinase, located in the cytoplasmic domain, triggering a complex array of enzymatic and biological events leading to cell proliferation and differentiation.
Overexpression of EGFR has been associated with many malignancies, including cancers of the stomach and colon. Evidence is accumulating which show that malignant as well as certain normal cells also produce other form(s) of EGFR. For example, A431 human epidermoid carcinoma cells have been shown to produce a truncated EGFR that encodes a 2.8 kb mRNA transcript and is thought to be the result of gene rearrangement in chromosome 7. Likewise, the normal rat liver produces a 2.7 kb mRNA transcript whose 5xe2x80x2, but not 3xe2x80x2, sequences show 100% homology with the external domain of the full-length rat EGFR. This and other relevant observations suggest that this truncated form of EGFR mRNA, whose protein product is also secreted from the cell, is generated from alternative splicing of the primary EGFR transcript. A shorter 1.8 kb alternative transcript from the human EGFR, which also produces a secreted product, has been isolated from a human placental cDNA library (Reiter J. L., Maihle, N. J. Nucleic Acid Research 24:4050-4056, 1996).
The present invention is directed toward a polynucleotide sequence, proteins transcribed from the nucleotide sequence, methods for the use of epidermal growth factor receptor related protein (ERRP) as well as probes for the detection of m-RNA, DNA and cDNA of the described nucleotide sequence and monoclonal antibodies directed toward ERRP.
In particular, a cDNA fragment clone of 1583 base pairs with 90-95% sequence homology to mouse epidermal growth factor receptor (EGFR) and a truncated rat EGFR was isolated. The full length cDNA revealed 1958 base pairs (SEQ ID NO: 1) that contained 227 base pairs of 5xe2x80x2 untranslated region and an open reading frame encoding 478 amino acids (SEQ. ID NO: 2) followed by 290 base pairs of an untranslated region. The full length cDNA showed an 84% and 91% homology, respectively, to a rat truncated EGFR and the mouse EGFR. The product of the newly isolated DNA is referred to as ERRP (EGF-Receptor Related Protein). In a Northern-blot analysis with poly A+ RNA from different rate tissues, ERRP cDNA hybridized strongly to a mRNA transcript of about 1.8 Kb. Maximal expression was noted in the small intestine, followed by colon, liver gastric mucosa and other tissue. Transfection of ERRP cDNA in HCT-116 cells, a colon cancer cell line, markedly inhibited (40-60%) proliferation in monolayer and soft agar and also attenuated EGFR tyrosine kinase activity compared to vector-transfected control cells. Proliferation of the vector-transfected control, but not ERRP transfected HCT cells could be stimulated by TGF-xcex1 (10xe2x88x9210M and 10xe2x88x929M). The over expression of ERRP in HCT-116 cell delayed tumor growth in SCID mice xenografts. The ERRP cDNA represents a new member of the EGFR gene family and the protein product plays a key role in modulating the function of EGFR.